Animal mus musculus hybrid cell clone l-producer of monoclonal cholera toxin antibodies

FIELD: medicine.

SUBSTANCE: there is produced a new CT-B1/F8 hybrid cell clone producing monoclonal cholera toxin antibody (MCAB) in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises MCAB specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:20000 in the cultural fluid, 1:2000000 in ascitic.

EFFECT: antibodies can be used for designing immunobiological systems of cholera toxin detection exceeding available analogues in sensitivity.

2 dwg, 4 ex

 

The invention relates to the field of Microbiology and biotechnology, specifically to hybridoma technology, and is a clone of the hybrid cells CT-B1/F8 animals Mus Musculus L producing cell cultures and peritoneal cavity of syngeneic animals monoclonal antibodies (hereinafter MCAT) to the V. cholerae toxin (XT). Hybridoma can be used in diagnostic test systems for the specific indication of the cholera toxin in the food industry, environmental protection, medicine.

Vibrio cholerae (V. cholerae) and the toxin produced by them are a cause of acute intestinal diseases of humans, often ending in death. The V. cholerae toxin completely determines the symptoms of cholera and is one of the Central objects of monitoring of bacterial toxins in the environment and food. Currently, the analysis of toxins of great importance in connection with the threat of bioterrorism, as many natural toxins, including cholera toxin, can be used as components of biological weapons. The dose of the toxin that causes the disease, as a rule, extremely small [Scarlatos, A., Welt, C., C. Cooper, et al. // J. Food. Sci. 2005. V.70 (8). P.121-124], and therefore the task of the laboratory diagnosis of toxins is to develop tests with the highest possible senses is lacking. XT should be tested at a concentration not exceeding 1 ng/ml [Ramamurthy T., Bhattacharya S.K., Uesaka y, Horigome, K., Paul M., Sen D., Pal, S.C., T. Takeda, Takeda Y, Nair G.B. // J. Clin. Environ. 1992. V.30 (7). P.1783-1786].

Now for fast and sensitive diagnosis of toxins is the most used and reliable systems are based on enzyme-linked immunosorbent assay (ELISA) using antibodies against toxins [The EFSA Journal. 2007, v.130. P.4-352].

The sensitivity and specificity of ELISA is determined largely by the quality of the antibodies. Antigenic similarity between representatives cholerophobia group of toxins, primarily this refers to the similarity between XT and thermolabile toxin E. coli (LT), dictates the feasibility of using MCAT with strict specificity to XT.

To determine toxins MKAT are commonly used in the competitive and sandwich options ELISA. In the competitive version of MCAT adsorb on a solid substrate, then add a solution of purified toxin conjugated to peroxidase, Biotin or fluorescent label, and the sample containing the toxin. The toxin contained in the sample compete for binding sites on MKAT with highly purified, and conjugated with accurate toxin. The concentration of the test toxin is determined by a calibration curve obtained in a competitive ELISA with a toxin known concentration of the. The sensitivity of the determination of XT in the competitive ELISA is 26 ng/ml [Rucker V., Havenstrite K.L., Herr, A.E. Antibody microarray for native toxin detection. // Anal. Biochem. 2005. V.339. P.262-270]. In the test used MCAT (firm "Biodesign International).

In the sandwich version of the toxin from solution associated MKAT, adsorbed on a solid substrate, then (the second layer) with toxin other contact detecting MCAT conjugated with a label (for example, peroxidase, Biotin or fluorescein). The concentration of the test toxin is determined by a calibration curve obtained in the sandwich variant of the ELISA toxin known concentration. The sensitivity of the determination of XT in the sandwich variant of ELISA reaches - 1 ng/ml [N. Koike engineering Germany, K. Okada, Y. Yabushita, D.Y. Zhang, K. Yamamoto, T. Miwatani, T. Honda. Rapid and differential detection of two analogous enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli by a modified enzyme-linked immunosorbent assay. // FEMS Immunol Med Environ. 1997, 17 (1): 21-25].

When using MCAT (firm "Biodesign International) in sandwich version immobilized on the liposomes by ganglioside GM1 - cell receptor XT and LT the detection sensitivity up to 10 FG/ml [Ann-S. Yoon, DeCory T.R., A.J. Baeumner, Durst R. / Anal Chem. Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin. 2003.75. 2256-2261]. The use of ganglioside GM1 limits the specificity of the assay, since GM1 binds both XT and LT.

There is a method for quantitative detection of biological toxins-based ELISA the use of biological microchips. Biological microchip is an ordered array of individual micro-cells on a solid substrate, in which immobilized MKAT to various biotoxins bacterial and plant origin. The use of microarrays allows you to simultaneously analyze the sample for the presence of several biotoxins with sensitivity, not inferior to the sensitivity of standard immunological tests. In the microchip using a competitive option the sandwich option ELISA. The detection sensitivity XT in biological chip reaches 1.6 ng/ml [F.S. Ligler, C. Rowe Taitt, L.C. Shriver-Lake, K.E. Sapsford, Ya Shubin and J.P. Golden. Array biosensor for detection of toxins. 2003. Anal Bioanal Chem 377 (3): 469-477]. Such sensitivity can be achieved using chip biosensor. Simultaneously analyze XT and four other toxin, namely the ricin, staphylococcal toxin SeB, botulinum toxin A and toxin of Bacillus globigii. The chip used sandwich option ELISA, in which the connecting MKAT to XT and four other toxins (ricin, staphylococcal toxin SeB, botulinum toxin A and toxin of Bacillus globigii) locally immobilized on the glass surface by formation of a complex of avidin-biotinylated antibodies. For detection XT and other toxins using fluorescein-labeled MKAT. Fluorescent signals recorded by a confocal microscope. For detection use XT M is at clone hybridoma 3D11 (firm "Biodesign International"), which do not have a strict specificity, and are associated with XT and LT.

Known hydrogel microchip, which is an ordered array of three-dimensional hydrogel cells on a solid substrate obtained by the method of photo - or chemically modified polymerization and containing immobilized MKAT to various biotoxins bacterial and plant origin [RF Patent N 2216547, C07K 17/08, publ. 2003]. Hydrogel microchip is designed for screening of a wide range of dangerous toxins which can be used as components of biological weapons. Currently, however, the chip detects a limited number of toxins and, in particular, will not detect XT, which poses a serious threat to the health and lives of people.

Known closest to the claimed hybrid clone of cultured animal cells Mus musculus L SCC (P) N 469D - producer of monoclonal antibodies to cholerea enterotoxin [RF Patent N1835848, MKI C12N 5/00, publ. 1995]producing monoclonal antibodies (N 469) class lgM obtained against chromatographically purified cholera toxin, when hybridization of splenocytes with myeloma cells X63.Ag8.653. MKAT N 469 contact with the B-subunit of cholera toxin. The main disadvantage of a clone producing MKAT N 469, is that with his participation in the sandwich variant of ELISA to detect olery toxin with high enough sensitivity - 1 ng/ml in Addition to clone N 469 characterized by a rather low productivity of the antibody titer in the culture fluid is 1:250-1:500, and in ascitic 1:1000000. Clone N 469 produces antibodies of class lgM, as a result is difficult to obtain pure antibody preparations, as to obtain pure drug lgM antibodies unsuitable standard method - chromatography on protein a-sepharose.

The objective of the invention is to obtain clones of hybrid cells producing high-affinity, MCAT against the cholera toxin, non-binding thermolabile toxin E. coli and other bacterial toxins, with a limit of detection of toxin in IFA below 1 ng/ml.

The problem is solved by hybrid clone of cultured animal cells Mus musculus L CT-B1/F8 - producer of monoclonal antibodies to cholerea toxin.

The specified clone hybrid of cultured animal cells Mus musculus L CT-B1/F8 obtained by fusion of myeloma cells lines SP-2/0 cells and lymph nodes of BALB/c mice immunized by cholera toxin (Sigma), when used as a melting agent, polyethylene glycol with molecular weight of 4000 and subsequent selection of hybrid cells on selective medium: medium Needle in the modification of Dulbecco with 20% serum fetal cow, 4 mm L-glutamine, 50 μm-mercaptoethanol and GAT (0,1 mm gipoksantin, 4×107M and what interina, of 1.6×105M thymidine). The method of limiting dilutions selected stable clones producing antibodies in indirect ELISA specific binding XT, and not connecting the LT. In the next stage of clones producing MKAT specific to XT, select the clones that sandwich variant of ELISA define XT concentration below 1 ng/ml Then select the clones that the microchip is not cross-interact with other bacterial toxins, namely with ricin, a lethal factor toxin of Bacillus anthracis, the protective antigen toxin of Bacillus anthracis, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, SeG. The selected clone CT-B1/F8 produces antibodies, which upon detection XT in the sandwich variant of ELISA allow to reveal XT in a concentration not exceeding 1 ng/ml In particular, the sensitivity of the sandwich variant of ELISA, where as developing antibodies used MKAT CT-B1/F8, and as binding antibodies are used MCAT, recognizing different antigenic determinant XT, is 0.2 ng/ml the sensitivity persists when determining the cholera toxin in water from open water, broth and milk.

MCAT can be used for the quantitative detection of the cholera toxin in a simultaneous and parallel analysis with a number of other biological toxins in the format of a biological microchip. Sensitivity determining the Oia in the format of the microchip reaches of 0.44 ng/ml

Hybridoma CT-B1/F8 characterized by the following properties.

1. Karyological characteristics

The karyotype of the cells of the clone CT-B1/F8 on species of mouse, the modal number of chromosomes 88.

2. Morphological characteristics.

Clone hybridoma CT-B1/F8 presents large rounded cells, similar in morphology to cells of the initial myeloma line SP-2/0.

Thandanani growing conditions in vitro

The seeding concentration when grown in vitro (1.0 to 2.0)×105CL/cm3. The environment of the cultivation medium Needle in the modification of Dulbecco with 10% serum fetal cows. The cultivation temperature (37°C). The content of CO2in the atmosphere of cultivation - 5%.

4. Cultural properties of clone

Hybridoma CT-B1/F8 is the suspension-monolayer, about 60% of the cells are in suspension, being attached to the surface of the culture vessel, the frequency of passage when sowing dose (1.0 to 2.0)×105CL/cm - 3-4 days. Hybrid clone not lost the ability to synthesize antibodies when passirovannye in vitro for 6 months (time of observation). Productivity clone antibodies in vitro is in terms of titer supernatants in ELISA 1:20000.

5. The cultivation of hybridoma in the animal body.

Species of animal is inbred mouse strain BALB/c. Cell dose of 1-2×106one mouse. 10-14 days before the introduction of hybridoma mice in RosNOU cavity injected with 0.5 ml of 2, 6, 10, 14 - tetramethylpentane (piers). The tumor grows mixed actinotaenium way. Ascites is formed on 7-12 day in the amount of from 3 to 10 ml per mouse. Prevelement 100%. Hybrid clone retains the ability to produce antibodies at a constant level (control antibody titer) when perejivanii animal cells for 6 passages (observation period). The titer of MCAT in ascitic fluids in ELISA is 1:2×106.

6. Contamination of the clone.

Contamination CT-B1/F8 bacteria, yeasts and fungi were not identified.

7. Biotechnology characteristic of a useful product.

Antibodies produced by cells of the clone CT-B1/F8, belong to the class of immunoglobulins lgGl according to ELISA with teruyuki the monospecific antisera against certain classes of immunoglobulins mouse. Light chain MKAT CT-B1/F8 belong to "K" type.

By immunoblotting shows that MKAT CT-B1/F8 associated with the B-subunit of cholera toxin.

The affinity constants calculated from Beatty [J.D. Beatty, Beatty B.G. and Vlahos G. W.: Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immnol Methods, 1987; 100: 173-179], is for MCAT CT-B1/F8 - 0,52×109M-1.

8. Method of cryopreservation.

Environment cryopreservation contains 90% serum fetal cows and 10% cryoprotectant is dimethyl sulfoxide (DMSO).

The mode of freezing and thawing.

Freezing: residue glue is OK, obtained after low-speed centrifugation, suspended in the medium for the cryopreservation of calculation of 1-3×106CL in ml of medium. Cell suspension is stirred and poured into 1 ml of cryoprobes (Nunc). Labeled test tubes are placed in a low temperature refrigerator (-70°C) at night, and then in the Dewar vessel with liquid nitrogen for long term storage). Recovery of cells after thawing: rapid thawing in a water bath at 37°C until complete thawing. Then the cell suspension is transferred into a sterile centrifuge tube with 10 ml of culture medium without serum. Cells precipitated by centrifugation (10 min at 150 g), then the sediment cells suspended in the medium Needle in the modification of Dulbecco containing 10% serum fetal cow and transfer to a container for culturing. Cell viability after cryopreservation is (80±10)%.

The invention is illustrated graphics.

Figure 1. Sandwich ELISA analysis of cholera toxin in PBS, milk, meat broth and water on the tablet using MKAT CT-B1/F8 as developing antibodies. The analysis of the obtained data the optical absorption (A 492 nm) calculated as the median of the optical absorption of three identical cells of the microplate. The analytical sensitivity of the assay, i.e., the lowest tested concentration CT, define CA is the concentration, corresponding to the value of optical absorption, of not less than two standard deviations, the optical absorption repeatedly measured zero point. The value of the sensitivity is determined experimentally, according to the obtained calibration curves and calculated as follows:

IF0+2·SD,

where IF0- the value of the fluorescence intensity for each dimension zero samples; SD - standard deviation from the mean value IF0.

SD is calculated by the following formula:

,

where- average fluorescence intensity in the measurement of the zero samples; n is the number of dimensions.

For curve presents the sensitivity is 0.20 ng/ml.

Figure 2. The calibration curves to determine the concentration ARTICLE in sandwich ELISA analysis on the microarray using MCAT CT-B1/F8 as developing antibodies.

The calculation of the intensity of the fluorescent signal from each cell of the biochip and the determination of concentrations of toxins is carried out with the help of special software ImagelAssay (MPI, Moscow). The analysis of the obtained data, the fluorescence intensity is calculated as the median signal from four identical gel cells. The value of the sensitivity is determined by the experts is mental, on the obtained calibration curve, and calculate, as indicated in the legend to figure 1. For curve presents the sensitivity is of 0.44 ng/ml

The invention is illustrated by examples

Example 1. Obtaining a clone CT-B1/F8.

Hybridoma get when you merge a mouse myeloma SP 2/0 cells of lymph nodes mouse BALB/c category SPF immunized with whole by cholera toxin (Sigma). On the first day and 14 day of immunization administered in the pads of the hind paws of mice at 10 µg/mouse cholera toxin diluted in 0.1 ml of physiological solution, with the addition of 0.1 ml of incomplete adjuvant's adjuvant. 6 days after the last immunization remove the popliteal lymph nodes and perform the hybridization of the 5×106lymphocytes of mice with 1×106cell myeloma line SP-2/0 1 ml of polyethylene glycol 4000 for 1 min joint incubation. After removal of the polyethylene glycol by centrifugation, the cells were seeded on 96-well plates at a layer of feeder cells-macrophages isolated from the peritoneal cavity of a mouse inbred lines. Selection of hybrid cells spend in the environment of a needle in a modification of Dulbecco, with 20% serum fetal cow, 4 mm L-glutamine, 50 μm-mercaptoethanol and GAT. After 21 days - time total loss of myeloma cells, cells hybridoma cultured on medium without aminopterin. Further cultivation of hybridomas is performed on the medium containing no selective components GAT. 3-fold cloning of hybridomas is performed by the method of limiting dilutions in 96-well tablets on a layer of feeder cells. In the last two klonirovania the percentage of positive clones is 100%.

For screening of antibody-producing clones producing MKAT, specifically linking XT, using indirect ELISA with XT. In the wells of ELISA-tablet absorb cholera toxin at a concentration of 1 μg protein/ml in 0.1 M bicarbonate buffer pH 9.0 in. In each well contribute 100 μl of antigen solution and incubated overnight at 4°C. After sorption of free binding sites blocked with 1% solution of bovine serum albumin at 37°C for 1 h, the Wells washed FSB with 0.1% tween-20, then make 100 μl/well analyzed supernatants selected from the wells, which grow and produce antibodies of hybridoma. Incubated for 1 h at 37°C. Washed, as described above, contribute 100 μl/well peroxidase conjugate rabbit antibodies against mouse IgG, incubated for 40 min at 37°C, washed, and contribute 100 μl/well of peroxidase substrate: solution of ortho-phenyldiamine at a concentration of 1 mg/ml in 50 mm citrate buffer, pH 4.5, containing 0.015% hydrogen peroxide. After development of the color reaction is stopped by adding 100 μl/well of 10% sulfuric acid. The colour intensity is recorded spectrophotometrically, is predelay the optical absorption at a wavelength of 492 nm. Selected clones producing MKAT, specifically linking XT.

For screening of antibody-producing clones producing MCAT, not interacting with thermolabile toxin E. coli, using an indirect ELISA with thermolabile toxin E. coli. The analysis is performed as with the XT, but in the wells of ELISA-tablet absorb thermo-labile toxin of E. coli at a concentration of 1 μg protein/ml in 0.1 M bicarbonate buffer pH 9.0 in. In each well contribute 100 μl of antigen solution and incubated overnight at 4°C. Selected clones producing MKAT, not interacting with thermolabile toxin E. coli.

Example 2. The use of MKAT CT-B1/F8 in the sandwich variant of ELISA for cholera toxin.

In the sandwich variant of ELISA using MKAT CT-B1/F8 as the detecting antibody, and as a linking - MKAT directed against epitopes of the molecule cholera toxin, which does not interact MCAT CT-B1/F8. Linking MCAT absorb the first layer at the bottom of the ELISA microplate, 100 μl/well at a concentration of 10 μg/ml of Free binding sites of the plastic block with 1%solution of dry milk for 1 hour at 37°C. Washed FSB with 0.1% tween-20. Then make test samples of 100 μl/well containing cholera toxin in various dilutions, incubated for 45 min at 37°C, washed FSB with 0.1% tween-20. Make conjugate CT-B1/F8 with Biotin in the amount of 100 μl/well at a concentration of na is Itel 10 µg/ml Incubate 45 min at 37°C, washed, make conjugate with streptavidin horseradish peroxidase, incubated (45 min at 37°C) and washed. Then make the peroxidase substrate: solution of ortho-phenyldiamine at a concentration of 1 mg/ml in 50 mm citrate buffer, pH 4.5, containing 0.015% hydrogen peroxide. After 15±5 min the reaction is stopped by adding 10% sulfuric acid. The colour intensity is recorded spectrophotometrically at a wavelength of 492 nm. Analysis of solutions with known concentrations of cholera toxin shows that the detection sensitivity XT reaches 0.2 ng/ml During incubation the cholera toxin with MKAT CT-B1/F8 not in the FSB, and 10% milk, broth, water from an open reservoir detection sensitivity XT unchanged at - 0.2 ng/ml (Fig 1.).

Example 3. Quantitative analysis of the cholera toxin using a biological microchip

On hydrogel chips immobilized linking MKAT (as in example 2) against the cholera toxin and MCAT against 8 other toxins (ricin, a lethal factor toxin of Bacillus anthracis, protective antigen toxin of Bacillus anthracis, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, SeG. The concentration of each antibody in the polymerization mixture is 0.8±0.2 mg/ml gel volume element of 0.1 nl. Each gel element contains one of the nine above-mentioned MKAT, in addition, the chip includes a control gel is the first elements, does not contain antibodies.

Add 30 ál of the test sample and 30 μl of the solution showing biotinylated monoclonal antibody CT-B1/F8 and incubated on the chip for 17 h at 37°C. After washing (20 min, the FSB with 0.1% tween-20) add fluorescently labeled streptavidin, incubated 10 min at 37°C, washed and re-register the fluorescent signals. The detection limit for XT is of 0.44 ng/ml

Hydrogel chips receive, as described, for example, in the Patent of Russian Federation N 2216547, MKI C07K 17/08, epubl. Glass slides for the manufacture of microchips process solutions 1 N. NaOH, H2SO4, washed with water, then immersed in a 1% solution of 3-methacryloxypropyltrimethoxysilane in ethanol, washed in ethanol, water and dried. The polymerization mixture containing the gel-forming monomers on the basis of methacrylamide and N-substituted amino sugars, and subject to immobilization MKAT, applied with the help of a robot QArray (Genetix, UK) in the form of droplets with a volume of 0.1 nl to the surface of the activated substrate. Polymerization of the gel cells are under the lamp ultraviolet light with a maximum emission at 350 nm (GTE Sylvania lamp, F15T8/350B1, UK) at a distance of 8 cm from the lamp, for 50 min at 20°C

in a stream of nitrogen. When applying PTS 150 μm get gel elements hemispherical di the meter 120 μm. Biochips after polymerization washed for 40 min FSB with 0.1% tween-20. To reduce nonspecific interaction of the reagent with the surface of the biochip surface treated with a blocking buffer for 1 h, then washed with distilled water.

The quality of the biochips check in transmitted light using a biochip analyzer (MPI RAS), equipped with special software TestChip and QualityControl (MPI RAS). In the quality assurance rejected biochips, for which the deviation of the values of the radii of gel elements exceed 5% within each biochip and 8% among all biochips of this party.

Example 4. Simultaneous analysis of several biotoxins, including the cholera toxin on a single microchip. The assay procedure is as in example 3, but biotinylation MKAT CT-B1/F8 detecting XT, contribute in a mixture with biotinylating antibodies to other toxins (ricin, a lethal factor toxin of Bacillus anthracis, the protective antigen toxin of Bacillus anthracis, diphtheria toxin, staphylococcal toxins SeA, SeB, Sel, SeG. The limits of detection of biotoxins for parallel analysis are the same as the definition of each toxin alone. For XT is minimal compared to a dose of 0.44 ng/ml

The proposed analysis can be used to test for the presence of toxin in food,in water and biomaterials.

Clone hybrid of cultured animal cells Mus musculus L ST-B1/F8 producing monoclonal antibodies to cholerea toxin.



 

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FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: cellular biology, medicine.

SUBSTANCE: invention relates to isolating and cryopreserving precursor-cells. Methods involve treatment of human liver tissue for preparing the essentially monocellular suspension containing precursor-cells and cells that are not precursor-cells, a single or more lines of cellular differentiation presenting in the human liver. Invention describes methods involving stage for separating cellular population resulting to reducing amount of cells that are not precursor-cells and providing preparing the separated suspension enriched with precursor-cells expressing one or more markers and associated with a single or more lines of the cellular differentiation. Also, invention describes a method for selection cells from the separated suspension wherein these cells or their progeny, or their more matured forms express one or more markers associated with lines of the cellular differentiation. These markers involve: CD14, CD34, CD38, CD45 and ICAM. Hepatic precursor-cells have diameter size 6-16 mc, they are diploid and show indices: glycoforin A-, CD45-, AFP+++, ALB+, ICAM+ and they comprise subpopulations varying with respect to expression of CD14+, CD34++, CD38++ and CD117++. These cells are useful for carrying out cellular and genetic therapy in liver treatment and for preparing artificial organs also.

EFFECT: valuable biological and medicinal properties of cells.

41 cl, 7 tbl, 13 dwg, 15 ex

FIELD: medicine, surgery, transplantology.

SUBSTANCE: embryonic spleen should be sampled, washed in nutritive medium № 199 to be placed into fresh medium № 199 to obtain homogenate in teflon homogenizer followed by centrifuging; then one should isolate the upper, medium and inferior layers, suck off medium layer and the upper part of inferior layer; the cell mixture obtained should be diluted in nutritive medium № 199 to be then introduced by injections into mesentery of small intestine or rectus muscle of abdomen. The present innovation favors the activation of immune system in patients undergone splenectomic operation and in those in case of surgical immunodefficient state due to high functional and regenerating activity of transferred embryonic splenic cells.

EFFECT: higher efficiency of prophylaxis.

6 dwg, 2 tbl

FIELD: medicine, genetic engineering.

SUBSTANCE: invention relates to applying genetic engineering approaches for treatment of autoimmune diseases, in particular, for treatment of cerebrospinal sclerosis. This is achieved by incorporation of one or some recombinant genes encoding autoantigens that represent a target for autoimmune response. In particular, invention claims a method for designation of gene encoding encephalitogenous epitope of proteolipid protein and expression of gene product in vivo by using the recombinant retroviral vector. Expression and secretion of encephalitogenous epitope improves histopathological and clinical indices in experimental autoimmune encephalomyelitis in mice that is used as a model of cerebrospinal sclerosis. The advantage of invention involves the development of a method for recovery the tolerance in treatment of cerebrospinal sclerosis being without suppression of immune system.

EFFECT: improved and valuable method for treatment.

6 cl, 13 dwg, 3 ex

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology and pharmaceutical industry.

SUBSTANCE: title operations are accomplished by following way. Three-dimensional culture of stromal cells is cultured in piston flow bioreactor, in particular being introduced in fibrous matrix incorporated into substrate, which is placed in container constituting a part of bioreactor piston. Stromal cells are grown until density 5 x 106 cell/cm3 substrate is attained, whereupon non-differentiated hemopoietic cells are either sowed directly into piston flow bioreactor or cultured in conditioned medium of stromal cells obtained by gathering medium from indicated flow bioreactor. Non-differentiated hemopoietic cells obtained by cultivation in presence of three-dimensional culture of stromal cells or their conditioned medium are transplanted to into recipient.

EFFECT: enabled growth of large amounts of stromal cells within a relatively small volume to provide longer maintenance of vital activity and reproduction of non-differentiated hemopoietic stem cells or precursor cells.

77 cl, 9 dwg, 3 tbl

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

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